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1.
Bioinformatics ; 19(18): 2343-50, 2003 Dec 12.
Article in English | MEDLINE | ID: mdl-14668217

ABSTRACT

MOTIVATION: A whole set of Expressed Sequence Tags (ESTs) from the Sf9 cell line of Spodoptera frugiperda is presented here for the first time. By this way we want to identify both conserved and specific genes of this pest species. We also expect from this analysis to find a class of protein sequences providing a tool to explore genomic features and phylogeny of Lepidoptera. RESULTS: The ESTs display both housekeeping as well as developmentally regulated genes, and a high percentage of sequences with unknown function. Among the identified ORFs, almost all ribosomal proteins (RPs) were found with high EST redundancy and hence sequence accuracy. The codon usage found among RP genes is in average surprisingly much less biased in Lepidoptera than in other organisms. Other Spodoptera genes also displayed a low bias, suggesting a general genome expression feature in this Lepidoptera. We also found that the L35A and L36 RP sequences, respectively, display 40 and 10 amino-acid insertions, both being present only in insects. Sequence analysis suggests that they are probably not subjected to a strong selective pressure and may be good phylogenetic markers for Lepidoptera. Most interestingly, the Lepidoptera sequences of 9 RP genes displayed a specific signature different from the canonical one. We conclude that the RP family allows valuable comparative genomics and phylogeny of Lepidoptera. AVAILABILITY: All EST sequence data are available from the private 'Spodo-Base' upon request.


Subject(s)
Expressed Sequence Tags , Gene Expression Profiling/methods , Ribosomal Proteins/genetics , Sequence Alignment/methods , Sequence Analysis, Protein/methods , Spodoptera/genetics , Abstracting and Indexing/methods , Animals , Bias , Cell Line , Codon/genetics , Evolution, Molecular , Information Storage and Retrieval/methods , Phylogeny , Reproducibility of Results , Ribosomal Proteins/metabolism , Sensitivity and Specificity , Sequence Homology, Amino Acid , Spodoptera/metabolism
2.
Virus Genes ; 22(3): 247-54, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11450942

ABSTRACT

Genetic differences between strains of a baculovirus are often limited to some restriction sites, short DNA deletions or absence of some nonessential genes. The recently coined bro gene family, represents a new major source of intraspecific variability. A comparison between two bro gene sets of Bombyx mori nucleopolyhedroviruses (NPV) shows that bro genes are distributed in three regions for the -T3 and -SC7 virus strains. In BmNPV T3, five bro genes are distributed in three genome locations, whereas the BmNPV SC7 strain possess a single bro copy in each region. In addition, each of the BmNPV SC7 bro genes belongs to one of the three subfamilies present in BmNPV T3. Analysis of bro copy sequences and of adjacent sequences suggests an active redistribution of sequences due to intraspecific recombination. The maintenance of one allele of each subfamily suggests that they play different roles in the viral cycle, and that they are essential.


Subject(s)
Bombyx/virology , Genes, Viral , Nucleopolyhedroviruses/genetics , Viral Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Bombyx/cytology , Cell Line , Molecular Sequence Data , Open Reading Frames , Restriction Mapping , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Viral Proteins/chemistry
3.
J Gen Virol ; 81(Pt 6): 1605-13, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10811945

ABSTRACT

The coding sequences of four overlapping polypeptides starting at four different in-frame AUG codons and co-terminating at the stop codon of the cap gene of Junonia coenia densovirus (JcDNV) were inserted under the control of the p10 promoter of Autographa californica nucleopolyhedrovirus (AcMNPV) to generate AcMNPV-VP1 (four polypeptides), AcMNPV-VP2 (three polypeptides), AcMNPV-VP3 (two polypeptides), and AcMNPV-VP4 (one polypeptide) recombinant viruses. In all cases, infection of Spodoptera frugiperda cells (Sf9) by each of the four recombinant viruses resulted in the production of virus-like particles (VLPs) 22-25 nm in diameter. The VLPs produced by the three recombinants AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-VP4 were abundant and contained three, two and one polypeptides, respectively. VP4, the shortest polypeptide, thus appears to be sufficient for assembly of VLPs morphologically similar to those formed with two to four polypeptides. The ratio of VPs did not appear to be critical for assembly of the particles. The polypeptide starting at the first AUG immediately downstream from the p10 promoter was always the most abundantly expressed in infected cells, regardless of the construct. In contrast, plaque-purified AcMNPV-VP1 recombinants were unstable and produced less than one-twentieth of the VLPs produced by the others. All VP transcripts started at the TAAG late motif of the p10 promoter and had a poly(A) tail 14 nt downstream of a poly(A) addition signal located 98 nucleotides downstream of the common stop codon. No significant transcription initiation inside the cap sequence of AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-PV4 was observed.


Subject(s)
Capsid/metabolism , Densovirus/physiology , Virus Assembly , 3' Untranslated Regions , 5' Untranslated Regions , Animals , Base Sequence , Binding Sites , Capsid/genetics , Capsid/isolation & purification , Capsid Proteins , Cell Line , Chromosome Mapping , Genes, Viral , Genetic Vectors , Genome, Viral , Molecular Sequence Data , Mutagenesis, Insertional , Nucleopolyhedroviruses , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spodoptera/cytology , Virion/physiology
4.
J Gen Virol ; 79 ( Pt 4): 931-5, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9568990

ABSTRACT

Autographa californica nucleopolyhedrovirus (AcMNPV) does not replicate in Bombyx mori cells (Bm5, BmN). We have shown previously that when a short DNA sequence within AcMNPV ORF95, which encodes the viral helicase P143, is replaced with the colinear region of B. mori nucleopolyhedrovirus (BmNPV), AcMNPV gains the ability to replicate in Bm5 cells. To determine the mutational events in the p143 gene required to allow AcMNPV replication in B. mori cells, AcMNPV recombinants produced in Sf9 cells were screened in vivo in B. mori larvae, which are more permissive to baculovirus infection than B. mori cell lines. Eight combinations of mutations were tested and characterization of viral DNA extracted from dead larvae showed that amino acid changes at position 564 and 577 are required to kill B. mori larvae.


Subject(s)
Bombyx/virology , Genes, Viral , Nucleopolyhedroviruses/genetics , Nucleopolyhedroviruses/pathogenicity , Point Mutation , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA Helicases/genetics , DNA Primers/genetics , DNA, Viral/genetics , Larva/virology , Molecular Sequence Data , Nucleopolyhedroviruses/physiology , Recombination, Genetic , Spodoptera , Transfection , Virulence/genetics , Virus Replication/genetics
5.
Virology ; 241(2): 276-84, 1998 Feb 15.
Article in English | MEDLINE | ID: mdl-9499802

ABSTRACT

Autographa californica nucleopolyhedrovirus (AcMNPV) lef-4 gene [ORF 90; Ayres et al. (1994) Virology 202, 586-605] is involved in both late and very late gene expression [Passarelli and Miller (1993) Virology 197, 704-714]. The transcriptional properties of this gene have been analyzed. It is transcribed as a single 1.6-kb mRNA and transcripts were first detected 3 h postinfection (pi). The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -56 from the translation start codon and +96 downstream of the stop codon. A rabbit polyclonal antiserum has been raised against an internal polypeptide of LEF-4. A 55-kDa protein was observed by Western blot analysis from 5 h pi. LEF-4 localizes preferentially in the nucleus of infected cells and is associated with the virogenic stroma.


Subject(s)
Gene Expression , Nucleopolyhedroviruses/genetics , Viral Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Western/methods , Cell Line , Chromosome Mapping , DNA, Viral , Microscopy, Immunoelectron , Molecular Sequence Data , Moths/virology , Nucleopolyhedroviruses/metabolism , Polymerase Chain Reaction , RNA, Messenger , RNA, Viral , Rabbits , Spodoptera/cytology , Subcellular Fractions/metabolism , Transcription, Genetic , Viral Proteins/biosynthesis , Viral Proteins/metabolism
6.
J Gen Virol ; 79 ( Pt 3): 629-37, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9519844

ABSTRACT

Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.


Subject(s)
Genes, Immediate-Early/genetics , Genes, Viral , Immediate-Early Proteins/genetics , Amino Acid Sequence , Animals , Baculoviridae/metabolism , Base Sequence , Cells, Cultured , Gene Expression Regulation , Insecta , Ligases/genetics , Methyltransferases/metabolism , Molecular Sequence Data , Occlusion Body Matrix Proteins , Polymerase Chain Reaction , Polynucleotide 5'-Hydroxyl-Kinase/genetics , Promoter Regions, Genetic , Sequence Homology, Amino Acid , Spodoptera , Transfection , Viral Fusion Proteins/metabolism , Viral Proteins/metabolism , Viral Structural Proteins
7.
J Invertebr Pathol ; 70(2): 143-9, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9281403

ABSTRACT

The S character of Drosophila simulans SimES-st strain undergoes a non-mendelian transmission. It has been postulated that a virus, Drosophila S virus (DSV), could be the causative agent. Electron microscopy analysis of gonads of flies showing a strong S phenotype revealed the presence of virus in or near the germ cells. The S character transmission rate is greater in females than that in males. Similarly, the level of infection by DSV is higher in ovaries than that in testes. Flies treated at a nonpermissive temperature do not present the S phenotype and appear to be cured from the virus. This information, taken together with previous work, makes the hypothesis that DSV is the causative agent of the S phenotype more than likely.


Subject(s)
Drosophila/virology , Insect Viruses/isolation & purification , Reoviridae/isolation & purification , Animals , Drosophila/ultrastructure , Female , Insect Viruses/ultrastructure , Male , Ovary/virology , Reoviridae/ultrastructure , Temperature , Testis/virology
9.
Virology ; 202(2): 586-605, 1994 Aug 01.
Article in English | MEDLINE | ID: mdl-8030224

ABSTRACT

The complete nucleotide sequence of the genome of clone 6 of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been determined. The molecule comprises 133,894 base pairs and has an overall A + T content of 59%. Our analysis suggests that the virus encodes some 154 methionine-initiated, and potentially expressed, open reading frames (ORFs) of 150 nucleotides or greater. These ORFs are distributed evenly throughout the virus genome on either strand. The ORFs are arranged as adjacent, nonoverlapping reading frames separated by short intergenic regions. Based on the primary nucleotide sequence, predictions have been made concerning the functions of certain genes, the sites for initiation of viral DNA replication, the regulation of early and late gene transcription, and factors that may affect the AcNPV gene translational efficiency. The genome sequence data confirm, with minor differences, the information obtained for other AcNPV clones. It is proposed that clone C6 is considered the archetype AcNPV for comparison purposes.


Subject(s)
DNA, Viral/genetics , Nucleopolyhedroviruses/genetics , Viral Proteins/genetics , Base Sequence , Codon , DNA, Circular/genetics , Genes, Viral , Molecular Sequence Data , Open Reading Frames , Repetitive Sequences, Nucleic Acid , Restriction Mapping , Viral Structural Proteins/genetics
10.
J Invertebr Pathol ; 59(3): 264-70, 1992 May.
Article in English | MEDLINE | ID: mdl-1607670

ABSTRACT

The expression of the S character of Drosophila simulans associated to hereditary reovirus DSV is affected by the rearing temperature. The transmission of the S character and the consequences of the length of high temperature (28 degrees C) treatments on that transmission were investigated. Larvae treated at 28 degrees C from eclosion to imaginal emergence produce few S individuals in the offspring. The offspring of larvae treated between the 144th hr of development (middle of the pupal stage) and the imaginal emergence show variable levels of S phenotype expression. These levels appear to be related to the age and phenotype of their mothers. Those facts are discussed under the hypothesis of S character being caused by the hereditary reovirus Drosophila S virus.


Subject(s)
Aging/genetics , Drosophila/genetics , Gene Expression , Analysis of Variance , Animals , Female , Male , Phenotype , Temperature
11.
Nature ; 352(6330): 85-8, 1991 Jul 04.
Article in English | MEDLINE | ID: mdl-2062383

ABSTRACT

Baculoviruses provide alternatives to chemicals for controlling insect pests and can be applied by spraying. Baculoviruses have a limited host range, but work relatively slowly. They are dissolved in the midgut of insect larvae to release infectious virions which enter gut epithelial cells and begin to replicate. Replication in other organs causes extensive tissue damage and eventually death. This process can take 4-5 days, but in the field may last for more than a week, allowing the larvae to feed for longer and thereby damaging the host plant. Baculovirus expression vectors expressing foreign genes, such as those for insect-specific toxins, hormones or enzymes, might alleviate this problem. We have now constructed a recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus containing an insect-specific neurotoxin from the venom of the North African (Algerian) scorpion, Androctonus australis Hector. The neurotoxin acts by causing specific modifications to the Na+ conductance of neurons, producing a presynaptic excitatory effect leading to paralysis and death; it has no effect in mice. Expression of the neurotoxin by the virus causes a reduction in the time required to kill the host insect.


Subject(s)
Baculoviridae/genetics , Insecticides , Neurotoxins/genetics , Pest Control, Biological , Recombination, Genetic , Amino Acid Sequence , Animals , Base Sequence , Gene Expression , Molecular Sequence Data , Neurotoxins/toxicity
12.
EMBO J ; 8(11): 3437-46, 1989 Nov.
Article in English | MEDLINE | ID: mdl-2510997

ABSTRACT

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. A permissive allele was cloned and sequenced. The structural gene (3.1 kbp) is divided into three exons. The mRNAs are heterogeneous in size. They differ only in the 5' end of the first exon. The sequence upstream of the short mRNAs contains classical promoter elements. No TATA and CAAT boxes are appropriately positioned upstream of the initiation sites of the long mRNAs, but several repeats, palindromic sequences and inverted CAAT boxes are present. These observations, together with the tissue-dependent distribution of short and long transcripts, support the hypothesis of the existence of at least two classes of genuine initiation sites. The long size of the untranslated leader RNA region suggests a control of gene expression at the translation level. The same translation product of 599 amino acids (76.3 kd) is predicted for all mRNAs, but the in vitro translation product migrates in SDS-PAGE with a higher apparent mol. wt (115-125 kd). The putative ref(2)P protein contains internal repeats, PEST regions which may be signals for protein degradation, and interesting structural motifs such as zinc finger and amphiphilic helices. These later motifs could be mitochondrial pre-sequences. The degeneration of mitochondria is observed in the spermatids of sterile male flies homozygous for the loss-of-function alleles. The amino acid sequence of the ref(2)P product shows no homology with any known protein from the data banks.


Subject(s)
Drosophila melanogaster/genetics , Genes , Rhabdoviridae/physiology , Alleles , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Drosophila melanogaster/microbiology , Electrophoresis, Polyacrylamide Gel , Exons , Fertility/genetics , Introns , Male , Molecular Sequence Data , Phenotype , Promoter Regions, Genetic , RNA, Messenger , Restriction Mapping , Transcription, Genetic , Virus Replication
13.
J Virol ; 63(2): 1007-9, 1989 Feb.
Article in English | MEDLINE | ID: mdl-2911113

ABSTRACT

The S character of Drosophila simulans, the absence or malformation or both of bristles and other cuticular structures, was described by Comendador (Drosophila Inf. Serv. 55:26-28, 1980). Its characteristics (maternal transmission, low pathogenicity, and sensitivity to temperature) suggested the existence of a virus as the causative agent. Indeed, reoviruslike particles were found in subcuticular cells of S individuals, and its association with S phenotypic expression was shown. This virus was called Drosophila S virus (DSV) (C. Louis, M. López-Ferber, N. Plus, G. Kuhl, and S. Baker, J. Virol. 62:1266-1270, 1988). We report here the purification and analysis of some properties of DSV particles, the morphology (spherical, 60 nm in diameter with an electron dense central core and less dense shell) and genome composition (double-stranded RNA divided into segments), which classify DSV as a new member of the family Reoviridae.


Subject(s)
Drosophila/microbiology , Reoviridae/classification , Animals , RNA, Double-Stranded/analysis , RNA, Viral/analysis , Reoviridae/genetics , Reoviridae/isolation & purification , Reoviridae/ultrastructure
14.
J Virol ; 62(4): 1266-70, 1988 Apr.
Article in English | MEDLINE | ID: mdl-3346947

ABSTRACT

Isometric reolike virions were found in all the examined Drosophila simulans flies from two strains (SimES-st and Israel-st) presenting the S phenotype, a maternally inherited morphological trait (abnormalities of bristles). Normal flies of both strains appeared virus-free. Virions were found in the cytoplasm of male and female gonads and epidermal cells, including the bristle-forming cells, which appeared disorganized. Steps of virogenesis were described. A positive correlation was demonstrated between expressivity of the S phenotype and degree of viral infection. This hereditary reolike virus seems to be responsible for the S character of D. simulans and was named DSV (Drosophila S virus).


Subject(s)
Drosophila/microbiology , Insect Viruses/ultrastructure , Reoviridae/ultrastructure , Virion/ultrastructure , Animals , Drosophila/genetics , Female , Male , Microscopy, Electron , Phenotype
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